Vaccine comprising lactobacillus strains for treating prostate inflammation and benign prostate hyperplasias

ABSTRACT

The invention relates to vaccines for treating prostate inflammation and benign prostate hyperplasias (stages I and II) comprising  Lactobacillus  strains in an inactivated form and carriers and/or excipients commonly used in vaccine preparations.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application that claims benefit under35 USC § 120 of co-pending U.S. application Ser. No. 14/735,637 filedJun. 10, 2015 which is a continuation of U.S. application Ser. No.13/049,344 filed Mar. 16, 2011 issued as U.S. Pat. No. 9,056,073 on Jun.16, 2015, which is a continuation application of U.S. application Ser.No. 12/077,780 filed Mar. 21, 2008 now abandoned, which is acontinuation application of U.S. application Ser. No. 10/130,823 filedSep. 16, 2002 now abandoned, which is a 35 U.S.C. § 371 National PhaseEntry Application of International Application No. PCT/H000/00122 filedNov. 23, 2000, which designates the U.S., and which claims benefit under35 U.S.C. § 119(a) of the Hungarian Provisional Application No. P9904408filed Nov. 25, 1999, the contents of each of which are incorporatedherein by reference in their entireties.

FIELD OF INVENTION

The present invention relates to a vaccine comprising Lactobacillusstrains useful in treating prostate inflammation and benign prostatehyperplasias (stages I and II).

BACKGROUND OF THE INVENTION

The pathogenecity of certain Lactobacillus strains has been reported in1938 [F. Marshall: Der Doderleinische Bacillus vaginalis alsEndokarditiserreger, Zentr. Bad Parasit. Kde. I. Abt. Orig., 141:153-159(1938); E. Biocca es A. Sepilli: Human infections caused bylactobacilli, J. Inf. Dis., 81:112-115 (1947); W. Sims: A pathogenicLactobacillus, J. Path. Bact, 87:99-105 (1964); B. Rosan and B. F.Hammond: Toxicity of Lactobacillus case/, J. Dent. Res., 44:783-787(1965); M. E. Sharpe, L. R. Hill and S. P. Lapage: Pathogeniclactobacilli, J. Med. Microbiol., 6, 281-286 (1973).

G. Wied reported in 1952 [Zbl. Bact., 160:413 (1952)] that certainLactobacillus strains show mucous membrane damaging activity. Rosan andHammond [1965, ibid.] reported that, with Lactobacillus strains stronglypathogenic to mice, intradermal inoculation of bacteria both in livingand in thermally inactivated states causes necrosis on the back ofrabbits.

K. Ujhelyi has found that necrosis can be induced also by Lactobacillusstrains cultivated from vagina. Based on his observation, it can bestated that the body of the bacterium contains a toxin which isresponsible for damaging the epithelia [Ujhelyi K. et al.: Role ofLactobacillus in urogenital inflammations and their treatment withvaccination, Symposium cum participatione internationalis de BiocenosiVaginae, Smolenie, 1983]. Certain strains, injected intradermally to theback of rabbits, cause necrosis of smaller or larger areas, while otherscause necrosis only in higher concentration or do not cause necrosis atall. K. Ujhelyi has found that rabbits can be immunized by vaccinationagainst the necrotic effect. He vaccinated rabbits intramuscularly withvaccine produced from certain Lactobacillus strains. Six weeks later, heintradermally administered cell-suspensions prepared from strains thathave been shown previously to be necrotic, and observed that necrosiswas not caused or was only caused in a lesser degree than in the case ofnon-vaccinated rabbits.

Furthermore, K. Ujhelyi has found that Trichomonas vaginalis contributesto the rise in vaginal pH by consuming lactic acid produced byLactobacillus strains in the vagina, thereby promoting theover-proliferation of Lactobacillus strains. Consequently toxin ispresent in higher concentration which, by damaging the mucous membrane,causes cell necrosis.

Furthermore, it is known that Lactobacillus strains, because of theirreceptor inhibiting and antibiotic activity as well as pH-modifyingeffect, are antagonistic to pyogenic microorganisms [Reddy et al.:Natural antibiotic activity of Lactobacillus, Dairy Prod. J. 18:15-22(1983); Salminen et al.: Lactic acid bacteria in the gut in normal anddisordered states, Dig. Dis., 10:227-238 (1992)].

Recently, it has been shown that Lactobacillus strains can bind directlyto T-lymphocytes since both the T-helper and T-killer cells havespecific receptors for Lactobacillus strains. Furthermore, Lactobacillusstrains promote the gamma-interferon production of the lymphocytes andthe cytotoxic activity of the natural killer cells [De Simone C., et aA:Enhancement of immune response of murine Peyer's pothes by a dietsupplemented with yoghurt, J. Immunopharmacol., 1:87-95 (1987)]. It hasbeen shown that Lactobacillus strains aspecifically increase theproduction of IgM and IgG [Blocksma et al.: Adjuvant activity oflactobacilli, different effects of viable and killed bacteria, Clin.Exp. Immunol., 37:367-373]. Additionally, under experimental conditions,Lactobacillus strains show antitumour and macrophage-activating activity[Kato I. et al.,: Antitumor activity of Lactobacillus case/in mice,Gann, 72:517-523 (1983); Oda M. et air. Antitumor polysaccharide fromLactobacillus sp., Agric Biol. Chem., 47:1623-1627 (1983)]. H. Ruttgershas found that immunostimulation by Lactobacillus strains causes asignificant long-lasting rise in secretory immunoglobulin level in thevagina [Bacterial vaginitis: Protection against infection and secretoryimmunoglobulin levels in the vagina after immunization therapy withGynatren, Gynecol. Obstet Invest, 26:240-249 (1988)].

Gjhelyi et al. [1983, \bid.] used parenterally administeredLactobacillus strains for aspecific immunostimulation and observed thatthe Lactobacillus strains used, in contrast to other aspecificimmunostimulation (e.g. by BCG, endotoxins etc.), show protective effectagainst certain bacterial toxins. This applies especially to toxicLactobacillus strains.

In trials carried out with vaccines (GYNEVAC®, GYNATREN®, SOLCOTRICHOVAC®) made of strains cultured by Ujhelyi it has been demonstratedthat immunostimulation by Lactobacillus strains, in contrast to othertherapeutic treatments, restores the biological balance of the vagina,normalizes the pH, decreases the number of pathogenic bacteria, andcontributes to the propagation of Ddderlein-flora (a mixed population ofLactobacillus strains capable of being cultivated from vaginalspecimens). It is an accepted fact that inflammatory diseases of thevagina caused by bacterial and Trichomonas infections can be cured inthis way more successfully than by other therapy and that suchinflammatory conditions are a major cause of premature births.Therefore, the frequency of premature births can also be decreased bysuch therapy [see e.g. in Genitalinfektion der Frau(SOLCOTRICOVAC/GYNATREN), Geburtsch. u. Frauenheilk, 44:311 (1984); E.Lazar, Gy. Varga, I. Institoris and K. Ujhelyi: Investigating thefactors, especially vaccination with lactobacilli, influencing thepremature births, in Kazincbarcika (in Hungarian), Magyar NoorvosokLapja (Journal of Hungarian Gynaecologists), 51:353-356 (1986); E.Lazar, Gy. Varga, I. Institoris and K. Ujhelyi: Decreasing the ratio ofneonates with small weight by lactobact vaccination of pregnant women(in Hungarian), Orvosi Hetilap (Physicians Weekly), 37:2263-2268 (1981),Riittgers, 1988, ibid., K. Ujhelyi, Gy. Philipp, Gy. Plank and V. Sagi:The Trichomonas syndrome I (in Hungarian), Magyar Noorvosok Lapja(Journal of Hungarian Gynaecologists), 36:433-442 (1973); Sharon et al,New England Journal, Dec. 28, 1995.].

More than 50% of men aged 50 or more suffer from prostate hyperplasiaand/or prostate inflammation. In spite of numerous known and utilizedtherapies, treatment is often unsuccessful. Taking into considerationthe known and generally accepted pathogenesis, it could not be supposedthat such diseases can be healed with vaccines comprising Lactobacillusstrains successfully.

The inventors of the present invention have, however, found thatconditions in the prostate are favorable to the proliferation ofLactobacillus strains and that pathogenic lactobacilli can often becultivated from patients suffering from chronic prostate inflammationand/or prostate hyperplasia. On this basis, therapeutic utilization ofvaccines comprising Lactobacillus strains for treating such patients hasbeen achieved.

DISCLOSURE OF THE INVENTION

The invention relates to vaccines for treating prostate inflammationsand benign prostate hyperplasias (stages I and II) comprisingLactobacillus strains in inactivated form and carriers and/or excipientscommonly used in vaccine preparations.

In another aspect, the invention relates to the use of Lactobacillusstrains for producing vaccines capable of treating prostate inflammationand benign prostate hyperplasias (stages I and II).

In a further aspect, the invention relates to the use of Lactobacillusstrains for treating patients suffering from prostate inflammation andbenign prostate hyperplasias (stages I and II).

Furthermore, the invention relates to a method of treating patientssuffering from prostate inflammation and benign prostate hyperplasias(stages I and II) comprising administering an effective dose of astrain-suspension of Lactobacillus strains intramuscularly to a patientin need of such treatment.

In an embodiment of the method of the invention, the strain-suspensionof Lactobacillus strains comprises a mixed population of the saidstrains in inactivated form.

The lactobacilli used in the vaccine of the invention are Lactobacillusstrains used in the above-said vaccines GYNEVAC®, GYNATREN® andSOLCOTRICHOVAC® that previously have been cultivated from womensuffering from gynecologic inflammations of bacterial origin. The singlecultivated strains can be use per se or in the form of a blend of thestrains.

The vaccine of the invention can be produced by methods commonly usedfor preparing vaccines. Advantageously, the cultivated strains arestored in lyophilized form, then, before use, they are propagated byculturing in Man-Rogosa-Sharpe medium at 45° C.

The composition of the said medium and the preparation method are setforth below.

To 2300 ml of sterile water the following components are addedsequentially, after dissolving the previously added component:

Bactotripton (Raenal) 30 g Lablemko (Reanal) 30 g K₂HPO₄ 6 g triammoniumcitrate 6 g sodium acetate 15 g glucose 30 g lactose 30 g maltose 9 gyeast extract (Reanal) 15 g Tween 80 3 ml Salt solution (composition seebelow) 15 ml

The obtained solution is adjusted to 3000 ml by the addition of sterilewater, filtered on G4 filter, bottles in smaller volumes and sterilizedat 121° C.

The composition of the above-said salt solution is as follows: 28.75 gof MgSO₄-7H₂O, 6 g of MnSO₄-2H₂O and 1.7 of FeSO₄—H₂O dissolved in 250ml of sterile water.

After culturing, the cells are harvested by centrifuging and aresuspended in physiological saline solution and treated withformaldehyde. The inactivated cells are harvested and resuspended inphysiological saline solution. The level of dilution is adjusted on thebasis of the protein content of the suspension. The protein content ofthe vaccine (suspension) of the invention is at least 0.08 mg/ml, andmay be up to 1 mg/ml or more, preferably from about 0.08 to about 0.32mg/ml, more preferably about 0.16 mg/ml.

The dosage of the vaccine of the invention and the frequency of theadministration depend on the conditions of the patient and the severityof the symptoms to be treated. The precise dose and frequency ofadministration should be specified by the practicing physician. Duringtreatment, it is advantageous if the vaccine is administeredintramuscularly in a volume of 1 ml, once a week for five weeks.

The following example is given for the purpose of illustration of theinvention without the intention of limiting of the scope claimed

EXAMPLE

Investigations were carried out with the vaccine of the invention byadministering same to patients with a diagnosis of prostate inflammationand/or prostate hyperplasias (stages I and II). The patients wereadministered intramuscularly 1 ml of a vaccine comprising Lactobacillusstrains of the invention once weekly for 5 weeks, without any othermedical treatment. The results of the control examination carried outafter this cure are summarized in the following Tables.

Number of the treated patients: 127Diagnosis: prostate hyperplasia stages I and II

Condition of the Time elapsed after the treatment patients 4 to 8 weeks2 to 4 months 6 months Healed  52 (40.94%) Worsening of 60% of 94Improved 47 (37.0%) the condition examined Unchanged 28 (22.0%) was notpatients were Worsened 0 observed in any symptom- free. of the patients.Number of the treated patients: 168Diagnosis: prostate inflammation

Condition of the Time elapsed after the treatment patients 4 to 8 weeks2 to 4 months 6 months Healed 76 (45.23%) Worsening of 70% of 79Improved 61 (36.31%) the condition examined Unchanged 31 (18.45%) wasnot patients were Worsened 0 observed in any symptom- free. of thepatients.

As can be seen in the above Tables, a significant ratio of the patientswere healed or their conditions improved essentially.

1. A process for treating a patient suffering from prostateinflammation, benign prostate hyperplasia stage I, or benign prostatehyperplasia stage II, said process comprising vaccinating the patientwith a vaccine, said vaccine containing an effective amount of a mixtureof Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillusfermentum in its inactivated form.
 2. A method of treating a patientsuffering from prostate inflammation, benign prostate hyperplasia stageI, or benign prostate hyperplasia stage II, said method comprisingvaccinating said patient intramuscularly with an effective dose of asuspension of a mixture of Lactobacillus plantarum, Lactobacillusparacasei and Lactobacillus fermentum in its inactivated form.
 3. Themethod of claim 2, wherein the patient is suffering from a benignhyperplasia stage I or II.
 4. The method of claim 2, wherein saidLactobacillus strains previously have been cultivated from womensuffering from gynecologic inflammations of bacterial origin.